The discovery of RNA interference (RNAi) has opened up an entirely new field of biology and medicine. The ability of RNAi to specifically silence target genes has yielded not only a new tool for basic research but also raised the concept of developing medicines based on RNAi. RNAi works through the targeting of mRNA via sequence-specific matches and results in degradation of target mRNA or its translational inhibition, leading to the loss of protein expression. This is pharmacologically achieved via the introduction of small 19-21 bp dsRNA molecules called small interfering RNA (siRNA). Since its discovery 10 years ago, siRNA has been widely investigated in vitro for its utility in treating various diseases, such as cancer, neurodegenerative and infectious diseases.
A major barrier to further development of siRNA has been the inability to effectively deliver siRNA in vivo due to the large molecular weight (for example, 13 kDa) and polyanionic nature (e.g. 40 negative phosphate charges). Naked siRNA does not freely cross the cell membrane. Furthermore, unmodified, naked siRNAs are relatively unstable in blood and serum, as they are rapidly degraded by endo- and exonucleases, meaning that they have short half-lives in vivo. Typically, chemical modifications can be introduced into the RNA duplex structure so as to enhance biological stability without adversely affecting gene-silencing activity. Alternatively, they can be formulated with a delivery system that not only enhances cell uptake but also affords biological stability. Several chemical modifications to the backbone, base, or sugar of the RNA have been employed to enhance siRNA stability and activity. However, delivery systems are still required to facilitate siRNA access to its intracellular sites of action.
Indeed, various delivery systems have been developed to enhance the uptake of siRNA into the target tissues after systemic administration. These include the use of polymers [1], lipids [2] or nanoparticles [3,4]. Most of these vectors are cationic to ensure efficient interaction of particles with negatively-charged siRNA nucleotides and to facilitate their cell entry. However, the ability of these cationic particles to deliver siRNA systemically is often poor due to rapid uptake by reticuloendothelial (RES) organs [5], thereby hindering the delivery of these particles to the site of interest. To overcome this problem, polyethylene glycol (PEG) has been used extensively in the formulation as it decreases RES uptake of these particles. This PEGylation also permits the accumulation of the particles in sites where defective vasculature is present, such as tumors, owing to the “Enhanced Permeability and Retention” phenomenon [6].
For lipid-based delivery vectors, various methods for formulating polynucleotide-loaded PEGylated particles have been reported to date, including post-insertion [7], reverse-phase evaporation [8], detergent dialysis [9] and ethanol dialysis [10]. However, most of these methods, though effective, require relatively complicated and lengthy formulation procedures with the resulting particles suspended in an aqueous state. This has led to long-term storage issues including aggregation and/or fusion of the particles, hydrolysis of the lipids, and instability of siRNA nucleotides in an aqueous environment. Moreover, these formulations are also prone to be affected by stresses occurring during transport, such as agitation or temperature fluctuation [11]. These problems, along with the significantly increased effort required for large-scale production of these particles using the existing formulation procedures will limit the widespread adoption of siRNA-containing lipid-based products in the clinics. Clearly, there is a need to develop relatively simple and effective method to formulate siRNA-loaded nanocarriers where the final product is also suitable for long-long term storage.
In the past two decades, several therapeutics based on nanosized particles in the range of 1-1,000 nm have been successfully introduced for the treatment of cancer, pain, and infectious disease. Hydrophilic bio-macromolecules (such as peptides or siRNA), usually exhibiting poor membrane permeability and high sensitivity to environmental conditions (heat, pH, enzymatic degradation) are considered adequate candidates for intracellular delivery by means of nanocarriers. Such nanocarriers can prolong the blood circulation time of these macromolecules which suffer from short physiological half lives, followed by a rapid clearance. However, the number of clinically relevant nanocarriers used for such a purpose is scarce, and major challenges still remain to be solved, especially for their efficient delivery via the parenteral route of administration. siRNAs represent a class of hydrophilic bio-macromolecules where the application of appropriate nanocarriers is most needed to exploit their full therapeutic potential.